Genetic variations in patient with Parry–Romberg syndrome

Parry–Romberg syndrome is a rare craniofacial disorder which is characterized by progressive facial atrophy. The etiology and pathogenesis of the disease are not known. Herein, we report the genetic variants in patient with this disease. A 25-year-old woman was diagnosed with Parry–Romberg syndrome according to her clinical manifestation, which presented with typical progressive unilateral facial soft tissue atrophy. Using peripheral blood samples, Whole exome sequencing (WES) was conducted on this patient and her parents. Variant loci of the genes were validated by Sanger sequencing in her twin sister who had no Parry–Romberg syndrome. Subsequently, we searched the GeneCards®: the Human Gene Database for variant genes, annotated them and analyzed their functions. The results of WES showed that 2 genes (MTOR, DHX37) were mutated, and the variant loci were MTOR: NM_004958.4: exon31: c.4487A>T: p.Q1496L and DHX37: NM_032656.4: exon17: c.2180C>T: p.T727M, respectively. However, the variant loci were also detected in her twin sister by Sanger sequencing. The Human Gene Database for variant genes shows that the two genes may be associated with craniomaxillofacial developmental abnormalities. Although MTOR and DHX37 genes were tested and found to have mutations in patient with Parry–Romberg syndrome, these variants may not directly determine the clinical phenotype. When studying clinical etiology, other factors, such as the environment, should also be taken into account.

www.nature.com/scientificreports/ that the study on the genetic aspects of the family lineage can provide some new molecular information about the disease in order to facilitate our further understanding of the disease mechanism.

Methods
Participants. This study was conducted in accordance with the guidelines of the Declaration of Helsinki for Human Research and was approved by the Ethics Committee of Shanghai Ninth People's Hospital Affiliated to Shanghai Jiaotong University School of Medicine. Informed consent was obtained from all participants, and their rights to privacy were preserved. A 25-year-old woman was diagnosed with Parry-Romberg syndrome according to her clinical manifestations, which presented with typical progressive unilateral facial soft tissue atrophy. Her parents and twin sister had normal craniomaxillofacial development. This patient, her parents and sister were enrolled in this study. We collected 2 ml of peripheral venous blood from these participants for later genetic testing analysis.
Whole-exome sequencing. Whole Functional analysis of mutant genes. We searched the GeneCards ® : the Human Gene Database (https:// www. genec ards. org/) for variant genes, annotated them and analyzed their functions. The analysis of genes mainly includes genomic locations, molecular function, phenotypes, subcellular locations, super-pathway, interacting proteins and biological process.
Ethical standards. All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2008 (5). Informed consent was obtained from all patients for being included in the study.

Results
This 25-year-old woman diagnosed with Parry-Romberg syndrome firstly presented with facial skin atrophy on one side at the age of 11 years. The symptoms of facial atrophy gradually worsened, during which various immune-related indicators, such as antinuclear antibodies, were tested and the results were not abnormal. The patient's other systems, such as heart and kidney, showed no significant abnormalities in the associated tests. In later stages, the patient developed facial atrophy of subcutaneous tissue, fat and muscle. The patient's facial symptoms progressed for 8-10 years before no further changes were evident. The genetic profile of the patient's family line is shown in Fig. 1A. In this family line, only the patient has the disease; the rest of the family, including the patient's twin sister, have a normal facial phenotype. The patient underwent two operations-autologous fat transplantation on the atrophic face, which resulted in some improvement in the atrophic face shape. Since the patient was concerned about the alar deformity, we performed local flap transfer to correct the alar deformity (Fig. 2).
The results of Whole-exome sequencing revealed that there were 2 variant loci in two genes, which were named MTOR and DHX37, respectively. The variant locus of MTOR was MTOR: NM_004958.4: exon31: c.4487A>T: p.Q1496L with dbSNP ID (rs1441021703). The variant locus of DHX37 was DHX37: NM_032656.4: exon17: c.2180C>T: p.T727M with dbSNP ID (rs371186622). The variant frequencies of these two loci in the population were 0.000054 and 0.000367, respectively. Both variant genes were heterozygous in the patient. The variant MTOR was heterozygous in the patient's father, while it was not detected in the patient's mother. Similarly, the variant DHX37 was heterozygous in the patient's mother, and it was not detected in her father. www.nature.com/scientificreports/ As for the Sanger sequencing results, we validated the two variants in the patient's twin sister. The results of the test revealed that the patient's twin sister was heterozygous for both genetic variants of the locus (Fig. 1B). In other words, the patient's sister had the same two genetic variants as the patient. However, the facial clinical phenotype of the patient's sister was normal.
By searching the database, we annotated the variant genes as well as performed functional analysis. MTOR is short for mechanistic target of rapamycin kinase, and the protein encoded by this gene belongs to the phosphatidylinositol kinase-related kinase family. DHX37 (DEAH-Box Helicase 37) encodes a DEAD box protein. DEAD box proteins are putative RNA helicases, which are characterized by the conserved motif Asp-Glu-Ala-Asp (DEAD). The genomic locations of MTOR and DHX37 are 1p36.22 and 12q24.31 ( Supplementary Fig. 1). Phenotypes from GWAS catalog for MTOR and DHX37 include 34 and 15 items, respectively. The associated phenotypes of these 2 genes are listed in Supplementary Table 1 and Supplementary Table 2. MTOR gene is widely expressed in various parts of the cell, and the top 3 subcellular locations for MTOR mainly include lysosome, cytosol and nucleus (Supplementary Fig. 2A). As to the subcellular locations for DHX37 gene, they are focused on the nucleus (Supplementary Fig. 2B). There are 91 super-pathways for MTOR gene, of which the top 5 ones are RET signaling, Transcription Receptor-mediated HIF regulation, mTOR signalling, GAB1 signalosome and CD28 co-stimulation (Supplementary Table 3). For DHX37 gene, there are only 2 associated super-pathways including rRNA processing in the nucleus and cytosol and Gene Expression (Supplementary Table 4).
We also analyzed the expression of the 2 genes in normal human tissues. The mRNA expression in normal human tissues from GTEx, Illumina, BioGPS, and SAGE for MTOR and DHX37 genes was shown in Fig. 3. The

Discussion
Using Whole exome sequencing and Sanger sequencing, we examined genetic variants in patient with Parry-Romberg syndrome for the first time in a patient's family line. The detected variant loci were validated in the patient's twin sister. Our results suggest a new line of research for the correlation between genetic variants in Parry-Romberg syndrome and clinical phenotypes, that is, the detected loci variants may not lead directly to the clinical phenotype. Parry-Romberg syndrome is a rare disease, the etiology and pathogenesis of which are not yet known 1,13 . There are few studies on genetic variants of the gene for Parry-Romberg syndrome 1,13 . Jenny et al. detected differentially expressed genes in patients' diseased tissues relative to normal subjects by second-generation sequencing of patient as well as normal human skin 12 . The sequencing results showed that there were 349 differentially up-regulated genes and 111 differentially down-regulated genes in the patient's lesioned tissue. Upregulated genes are enriched in various pathways including nuclear factor-κB in response to tumor necrosis factor, inflammation mediated by chemokines, cell proliferation and immune responses. The most highly up-regulated gene was IL24, which a member of the interleukin 10 family of cytokines. This gene is strongly associated with inflammatory and autoimmune diseases. Many down-regulated genes are evolutionarily conserved and implicated in orofacial development. The top 3 genes with the most significant down-regulation folds were OPN1MW2, TRIM48, and C10orf96, respectively. Jenny et al. also conducted an interesting study, in which they performed microvascular free tissue transfer to correct contour deformity in patient with Parry-Romberg syndrome. They found that gene expression in patients 6 months after surgery produced a large number of differential genes compared to presurgery, and that differential gene expression was significantly less in post-surgery patients compared to healthy controls than in pre-surgery. This result suggests that there is a tendency for patients' diseased tissues to shift toward normal tissues after surgery. Lyon et al. conducted whole exome sequencing of skin from the patients of Parry Romberg disease. Candidate genes were filtered using human phenotype ontology (HPO) terms specific to the Parry Romberg phenotype 14 . The results indicated that Candidate genes which may be implicated include TCOF1, COL1A1 and COL4A2 14 . All of these studies provide valuable evidence for the development of genetic variation in hemifacial atrophy. www.nature.com/scientificreports/ Most patients with hemifacial atrophy are sporadic, and few patients with the same disease in the same family have been reported. But Anderson reported two cases of hemifacial atrophy in the same family, one in a 9-year-old boy and the other in his cousin, a 14-year-old girl 15 . So it is not known whether hemifacial atrophy runs in families. The relationship between hemifacial atrophy and localized scleroderma is still controversial. There are common characteristics as well as differences between them. Lewkonia reported a case of hemifacial atrophy with localized scleroderma of the legs and trunk, and the serum in his system is positive for antinuclear antibodies 16 . These findings support the possibility that the disease is a variant of localized scleroderma rather than a developmental abnormality.
In this current surgery, two variant loci in two genes, which were named MTOR and DHX37, were detected and verified. Our conventional wisdom might suggest that the detected variant loci are usually directly responsible for the clinical phenotype of the patient. However, we performed Sanger validation on the patient's clinically phenotypically normal twin sister and found that the patient's sister also had consistent genetic variants. This suggests that the occurrence of the genetic variants may not necessarily lead to the development of an abnormal clinical phenotype in patients with Parry-Romberg syndrome. We searched the database for the mutated genes and analyzed their functions. We found high transcript levels of both genes in tissues such as skin and skeletal muscle, and the higher transcript levels in the immune system should also be of interest. At the protein translation level, we found that both 2 genes had high translation levels in the blood and immune system. These results can partially explain the atrophy of the patient's facial skin tissue, muscle tissue, etc. In addition, previous studies have suggested that the disease is an autoimmune disorder, while the results of the present study suggest a strong correlation between the mutated genes and the immune system, which is also consistent with previous studies.
The present study also has some limitations. First, this study included only one family member with hemifacial atrophy disease, which is a modest contribution to the study exploring the genetic type of the disease. The inclusion of more family lines could provide more basis for genetic type studies, and the inclusion of more family lines would allow more specimens to be collected for testing as a way to discover more and more precise information about genetic variants. However, because the disease is a rare disease, inclusion of more family lines does present a greater difficulty. In addition, we did not validate the patient's parents in the Sanger sequencing validation, which may reduce the confidence of the variant gene detection. However, the Sanger validation of the patient's sister could partially compensate for this aspect. Finally, we were not able to collect lesioned tissue specimens from patients, and further testing of lesioned tissue specimens for variant gene expression proteins could also increase the precision of this study.

Conclusions
In the present study, two novel loci of MTOR and DHX37 genes were detected in a family of patient with Parry-Romberg syndrome, which were not reported in previous studies. These two genes have a strong correlation with the immune system, which is consistent with previous reports that the disease is an autoimmune disorder. The patient's twin sister had the same genetic locus variants but did not exhibit the abnormal clinical trait of hemifacial atrophy, which suggests these variants may not directly determine the clinical phenotype. When studying clinical etiology, other factors, such as the environment, should also be taken into account. www.nature.com/scientificreports/